Chemical methods for producing tagged nucleotides

Abstract

This disclosure provides systems and methods for attaching nanopore-detectable tags to nucleotides. The disclosure also provides methods for sequencing nucleic acids using the disclosed tagged nucleotides.

What is claimed is: 
     
       1. A method for determining the nucleotide sequence of a single-stranded nucleic acid comprising:
 (a) contacting the single-stranded nucleic acid, wherein the single-stranded nucleic acid is in an electrolyte solution which is in contact with a nanopore in a membrane and wherein the single-stranded nucleic acid has a primer hybridized to a portion thereof, with a nucleic acid polymerase and a tagged nucleotide under conditions permitting the nucleic acid polymerase to catalyze incorporation of the tagged nucleotide into the primer if it is complementary to the nucleotide residue of the single-stranded nucleic acid which is immediately 5′ to a nucleotide residue of the single-stranded nucleic acid hybridized to the 3′ terminal nucleotide residue of the primer, so as to form a nucleic acid extension product, wherein the tagged nucleotide comprises a poly-phosphate moiety having a terminal phosphate, a base which is adenine, guanine, cytosine, thymine, or uracil, or a derivative of each thereof, and a tag covalently coupled to the terminal phosphate of the nucleotide by a triazole, a 1,2-diazine, a disulfide, a secondary amine, a hydrazone, a thio-acetamide, or a maleimide-thioadduct; 
 wherein incorporation of a tagged nucleotide results in release of a polyphosphate having the tag attached thereto; and if a tagged nucleotide is not incorporated, iteratively repeating the contacting with a different tagged nucleotide until a tagged nucleotide is incorporated, with the proviso that (1) the type of base in each tagged nucleotide is different from the type of base in each of the other three tagged nucleotides, and (2) either the number of phosphates in the poly-phosphate moiety of each tagged nucleotide is different from the number of phosphates in the poly-phosphate moiety of the other three tagged nucleotides, or the number of phosphates in the poly-phosphate moiety of each tagged nucleotide is the same and the type of tag on each tagged nucleotide is different from the type of tag on each of the other three tagged nucleotides, 
 (b) determining if the tagged nucleotide has been incorporated into the primer to form a nucleic acid extension product in step (a) by applying a voltage across the membrane and measuring an electronic change across the nanopore resulting from the polyphosphate having the tag attached thereto generated in step (a) entering into, becoming positioned in, and/or translocating through the nanopore, wherein the electronic change is different for each different number of phosphates in the poly-phosphate moiety, or for each different type of tag, as appropriate, thereby identifying the nucleotide residue in the single-stranded nucleic acid complementary to the incorporated tagged nucleotide; and 
 (c) iteratively performing steps (a) and (b) for each nucleotide residue of the single-stranded nucleic acid being sequenced, wherein in each iteration of step (a) the tagged nucleotide is incorporated into the nucleic acid extension product resulting from the previous iteration of step (a) if it is complementary to the nucleotide residue of the single-stranded nucleic acid which is immediately 5′ to a nucleotide residue of the single-stranded nucleic acid hybridized to the 3′ terminal nucleotide residue of the nucleic acid extension product, 
 
       thereby determining the nucleotide sequence of the single-stranded nucleic acid;
 wherein in each tagged nucleotide:
 (i) the poly-phosphate moiety is at the 5′-position of the nucleotide; 
 (ii) the poly-phosphate moiety comprises 6 phosphates; and 
 (iii) the tag comprises an oligonucleotide and the 5′-end or the 3′-end of the oligonucleotide is covalently coupled to the terminal phosphate of the poly-phosphate moiety. 
 
 
     
     
       2. The method of  claim 1 , wherein the nucleic acid is DNA and the nucleic acid polymerase is a DNA polymerase, or wherein the nucleic acid is RNA and the nucleic acid polymerase is a reverse transcriptase. 
     
     
       3. The method of  claim 1 , wherein the number of phosphates in the poly-phosphate moiety of each tagged nucleotide is the same and the type of tag on each tagged nucleotide is different from the type of tag on each of the other three tagged nucleotides. 
     
     
       4. The method of  claim 1 , wherein each tag is covalently coupled to the terminal phosphate by a triazole, and each triazole is formed by a reaction between an azide and an alkyne. 
     
     
       5. The method of  claim 1 , wherein each triazole has the structure: 
       
         
           
           
               
               
           
         
         wherein R 1  comprises the tag, and R 2  comprises the nucleotide; or 
         wherein R 1  comprises the nucleotide, and R 2  comprises the tag. 
       
     
     
       6. The method of  claim 1 , wherein each tag is selected from the group consisting of the tags listed in Table 4 or 5, or wherein each tag comprises a chemical modification selected from the group consisting of the chemical modifications listed in Table 6, or wherein each tagged nucleotide is selected from the group consisting of the tagged nucleotides listed in Table 4. 
     
     
       7. The method of  claim 1 , wherein the oligonucleotide comprises an unnatural nucleotide comprising a group selected from the groups consisting of an L-nucleotide, a 2′,5′-linkage, an α-D-nucleotide, a non-naturally occurring internucleotide linkage, a non-naturally occurring base, a non-naturally occurring sugar moiety, an abasic unit, a chemical modification selected from the group consisting of the chemical modifications listed in table 6, and any combination thereof. 
     
     
       8. The method of  claim 7 , wherein the oligonucleotide comprises at least 30 monomer units. 
     
     
       9. The method of  claim 1 , wherein the oligonucleotide comprises an unnatural nucleotide comprising a group selected from the groups consisting of a non-naturally occurring base, a non-naturally occurring sugar moiety, an abasic unit, a chemical modification selected from the group consisting of the chemical modifications listed in table 6, and any combination thereof. 
     
     
       10. The method of  claim 9 , wherein the oligonucleotide comprises an unnatural nucleotide comprising a non-naturally occurring internucleotide linkage. 
     
     
       11. The method of  claim 10 , wherein the non-naturally occurring internucleotide linkage is a phosphotriester or thiophosphate diester. 
     
     
       12. The method of  claim 10 , wherein the oligonucleotide comprises an unnatural nucleotide comprising a spacer moiety comprising an alkyl group of at least 2 carbons to about 12 carbons. 
     
     
       13. The method of  claim 12 , wherein the oligonucleotide comprises a chemical modification at its 3′-terminus that protects it from exonuclease degradation. 
     
     
       14. The method of  claim 13 , wherein the chemical modification is selected from the group consisting of phosphorylation and covalent coupling with C 3 -alkyl to C 12 -alkyl spacers having terminal hydroxyl groups. 
     
     
       15. The method of  claim 14 , wherein at least one tag is represented by SEQ ID NOS. 16, 19-21, 24, or 34, or wherein at least one of the tagged nucleotides is selected from the group consisting of dT6P-T 4 -dSp 10 -T 16 -C6, dA6P-T 4 -Sp18-T 22 -C 3 , dA6P-T 4 -Sp18 2 -T 19 -C 3 , dA6P-T 4 -Sp9 2 -T 22 C 3 , dA6P-dT 6 -dTNH 6 -dT 18 -C 3 , and dT6P-T 6 -dSp 8 -T 16 -C 3 . 
     
     
       16. The method of  claim 1 , wherein the 3′-end of the oligonucleotide is covalently coupled to the terminal phosphate of a poly-phosphate moiety. 
     
     
       17. The method of  claim 16 , wherein the oligonucleotide comprises a chemical modification at its 5′ terminus that protects it from exonuclease degradation. 
     
     
       18. The method of  claim 17 , wherein the chemical modification at its 5′ terminus is selected from phosphorylation, and covalent coupling with a C 3 -alkyl to C 12 -alkyl spacers. 
     
     
       19. The method of  claim 17 , wherein the oligonucleotide comprises a linker comprising a cyanine dye moiety, or a cyanine dye moiety which is a Cy3 moiety. 
     
     
       20. The method of  claim 19 , wherein the oligonucleotide and/or linker comprises a spacer moiety comprising an alkyl group of at least 2 carbons to about 12 carbons.